6-substituted 2-quinolinones and 2-quinoxalinones as poly(adp-ribose) polymerase  inhibitors

ABSTRACT

The present invention provides compounds of formula (I), their use as PARP inhibitors as well as pharmaceutical compositions comprising said compounds of formula (I) 
     
       
         
         
             
             
         
       
     
     wherein n, R 1 , R 2 , R 3 , R 4  and X have defined meanings.

FIELD OF THE INVENTION

The present invention relates to inhibitors of PARP and providescompounds and compositions containing the disclosed compounds. Moreover,the present invention provides methods of using the disclosed PARPinhibitors for instance as a medicine.

BACKGROUND OF THE INVENTION

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is a member ofthe PARP enzyme family consisting of PARP-1 and several recentlyidentified novel poly(ADP-ribosylating) enzymes. PARP is also referredto as poly(adenosine 5′-diphospho-ribose) polymerase or PARS(poly(ADP-ribose) synthetase).

PARP-1 is a major nuclear protein of 116 kDa consisting of threedomains: the N-terminal DNA binding domain containing two zinc fingers,the automodification domain and the C-terminal catalytic domain. It ispresent in almost all eukaryotes. The enzyme synthesizespoly(ADP-ribose), a branched polymer that can consist of over 200ADP-ribose units. The protein acceptors of poly(ADP-ribose) are directlyor indirectly involved in maintaining DNA integrity. They includehistones, topoisomerases, DNA and RNA polymerases, DNA ligases, andCa²⁺- and Mg²⁺-dependent endonucleases. PARP protein is expressed at ahigh level in many tissues, most notably in the immune system, heart,brain and germ-line cells. Under normal physiological conditions, thereis minimal PARP activity. However, DNA damage causes an immediateactivation of PARP by up to 500-fold.

Among the many functions attributed to PARP, and especially PARP-1, isits major role in facilitating DNA repair by ADP-ribosylation andtherefore co-ordinating a number of DNA repair proteins. As a result ofPARP activation, NAD⁺ levels significantly decline. Extensive PARPactivation leads to severe depletion of NAD⁺ in cells suffering frommassive DNA damage. The short half-life of poly(ADP-ribose) results in arapid turnover rate. Once poly(ADP-ribose) is formed, it is quicklydegraded by the constitutively active poly(ADP-ribose) glycohydrolase(PARG), together with phosphodiesterase and (ADP-ribose) protein lyase.PARP and PARG form a cycle that converts a large amount of NAD⁺ toADP-ribose. In less than an hour, over-stimulation of PARP can cause adrop of NAD⁺ and ATP to less than 20% of the normal level. Such ascenario is especially detrimental during ischaemia when deprivation ofoxygen has already drastically compromised cellular energy output.Subsequent free radical production during reperfusion is assumed to be amajor cause of tissue damage. Part of the ATP drop, which is typical inmany organs during ischaemia and reperfusion, could be linked to NAD⁺depletion due to poly(ADP-ribose) turnover. Thus, PARP or PARGinhibition is expected to preserve the cellular energy level therebypotentiating the survival of ischaemic tissues after insult.

Poly(ADP-ribose) synthesis is also involved in the induced expression ofa number of genes essential for inflammatory response. PARP inhibitorssuppress production of inducible nitric oxide synthase (iNOS) inmacrophages, P-type selectin and intercellular adhesion molecule-1(ICAM-1) in endothelial cells. Such activity underlies the stronganti-inflammation effects exhibited by PARP inhibitors. PARP inhibitionis able to reduce necrosis by preventing translocation and infiltrationof neutrophils to the injured tissues.

PARP is activated by damaged DNA fragments and, once activated,catalyzes the attachment of up to 100 ADP-ribose units to a variety ofnuclear proteins, including histones and PARP itself. During majorcellular stresses the extensive activation of PARP can rapidly lead tocell damage or death through depletion of energy stores. As fourmolecules of ATP are consumed for every molecule of NAD⁺ regenerated,NAD⁺ is depleted by massive PARP activation, in the efforts tore-synthesize NAD⁺, ATP may also become depleted.

It has been reported that PARP activation plays a key role in both NMDA-and NO-induced neurotoxicity. This has been demonstrated in corticalcultures and in hippocampal slices wherein prevention of toxicity isdirectly correlated to PARP inhibition potency. The potential role ofPARP inhibitors in treating neurodegenerative diseases and head traumahas thus been recognized even if the exact mechanism of action has notyet been elucidated.

Similarly, it has been demonstrated that single injections of PARPinhibitors have reduced the infarct size caused by ischemia andreperfusion of the heart or skeletal muscle in rabbits. In thesestudies, a single injection of 3-amino-benzamide (10 mg/kg), either oneminute before occlusion or one minute before reperfusion, caused similarreductions in infarct size in the heart (32-42%) while1,5-dihydroxyisoquinoline (1 mg/kg), another PARP inhibitor, reducedinfarct size by a comparable degree (38-48%) These results make itreasonable to assume that PARP inhibitors could salvage previouslyischaemic heart or reperfusion injury of skeletal muscle tissue.

PARP activation can also be used as a measure of damage followingneurotoxic insults resulting from exposure to any of the followinginducers like glutamate (via NMDA receptor stimulation), reactive oxygenintermediates, amyloid β-protein,N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its activemetabolite N-methyl-4 phenylpyridine (MPP⁺), which participate inpathological conditions such as stroke, Alzheimer's disease andParkinson's disease. Other studies have continued to explore the role ofPARP activation in cerebellar granule cells in vitro and in MPTPneurotoxicity. Excessive neural exposure to glutamate, which serves asthe predominate central nervous system neurotransmitter and acts uponthe N-methyl D-aspartate (NMDA) receptors and other subtype receptors,most often occurs as a result of stroke or other neurodegenerativeprocesses. Oxygen deprived neurons release glutamate in great quantitiesduring ischaemic brain insult such as during a stroke or heart attack.This excess release of glutamate in turn causes over-stimulation(excitotoxicity) of N-methyl-D-aspartate (NMDA), AMPA, Kainate and MGRreceptors, which open ion channels and permit uncontrolled ion flow(e.g., Ca²⁺ and Na⁺ into the cells and K⁺ out of the cells) leading tooverstimulation of the neurons. The over-stimulated neurons secrete moreglutamate, creating a feedback loop or domino effect which ultimatelyresults in cell damage or death via the production of proteases, lipasesand free radicals. Excessive activation of glutamate receptors has beenimplicated in various neurological diseases and conditions includingepilepsy, stroke, Alzheimer's disease, Parkinson's disease, AmyotrophicLateral Sclerosis (ALS), Huntington's disease, schizophrenia, chronicpain, ischemia and neuronal loss following hypoxia, hypoglycemia,ischemia, trauma, and nervous insult. Glutamate exposure and stimulationhas also been implicated as a basis for compulsive disorders,particularly drug dependence. Evidence includes findings in many animalspecies, as well as in cerebral cortical cultures treated with glutamateor NMDA, that glutamate receptor antagonists (i.e., compounds whichblock glutamate from binding to or activating its receptor) block neuraldamage following vascular stroke. Attempts to prevent excitotoxicity byblocking NMDA, AMPA, Kainate and MGR receptors have proven difficultbecause each receptor has multiple sites to which glutamate may bind andhence finding an effective mix of antagonists or universal antagonist toprevent binding of glutamate to all of the receptor and allow testing ofthis theory, has been difficult. Moreover, many of the compositions thatare effective in blocking the receptors are also toxic to animals. Assuch, there is presently no known effective treatment for glutamateabnormalities.

The stimulation of NMDA receptors by glutamate, for example, activatesthe enzyme neuronal nitric oxide synthase (nNOS), leading to theformation of nitric oxide (NO), which also mediates neurotoxicity. NMDAneurotoxicity may be prevented by treatment with nitric oxide synthase(NOS) inhibitors or through targeted genetic disruption of nNOS invitro.

Another use for PARP inhibitors is the treatment of peripheral nerveinjuries, and the resultant pathological pain syndrome known asneuropathic pain, such as that induced by chronic constriction injury(CCI) of the common sciatic nerve and in which transsynaptic alterationof spinal cord dorsal horn characterized by hyperchromatosis ofcytoplasm and nucleoplasm (so-called “dark” neurons) occurs.

Evidence also exists that PARP inhibitors are useful for treatinginflammatory bowel disorders, such as colitis. Specifically, colitis wasinduced in rats by intraluminal administration of the haptentrinitrobenzene sulfonic acid in 50% ethanol. Treated rats received3-aminobenzamide, a specific inhibitor of PARP activity. Inhibition ofPARP activity reduced the inflammatory response and restored themorphology and the energetic status of the distal colon.

Further evidence suggests that PARP inhibitors are useful for treatingarthritis. Further, PARP inhibitors appear to be useful for treatingdiabetes. PARP inhibitors have been shown to be useful for treatingendotoxic shock or septic shock.

PARP inhibitors have also been used to extend the lifespan andproliferative capacity of cells including treatment of diseases such asskin aging, Alzheimer's disease, atherosclerosis, osteoarthritis,osteoporosis, muscular dystrophy, degenerative diseases of skeletalmuscle involving replicative senescence, age-related musculardegeneration, immune senescence, AIDS, and other immune senescencedisease; and to alter gene expression of senescent cells.

It is also known that PARP inhibitors, such as 3-amino benzamide, affectoverall DNA repair in response, for example, to hydrogen peroxide orionizing radiation.

The pivotal role of PARP in the repair of DNA strand breaks is wellestablished, especially when caused directly by ionizing radiation or,indirectly after enzymatic repair of DNA lesions induced by methylatingagents, topoisomerases I inhibitors and other chemotherapeutic agents ascisplatin and bleomycin. A variety of studies using “knockout” mice,trans-dominant inhibition models (over-expression of the DNA-bindingdomain), antisense and small molecular weight inhibitors havedemonstrated the role of PARP in repair and cell survival afterinduction of DNA damage. The inhibition of PARP enzymatic activityshould lead to an enhanced sensitivity of the tumor cells towards DNAdamaging treatments.

PARP inhibitors have been reported to be effective in radiosensitizing(hypoxic) tumor cells and effective in preventing tumor cells fromrecovering from potentially lethal and sublethal damage of DNA afterradiation therapy, presumably by their ability to prevent DNA strandbreak rejoining and by affecting several DNA damage signaling pathways.

PARP inhibitors have been used to treat cancer. In addition, U.S. Pat.No. 5,177,075 discusses several isoquinolines used for enhancing thelethal effects of ionizing radiation or chemotherapeutic agents on tumorcells. Weltin et al., “Effect of 6(5-Phenanthridinone, an Inhibitor ofPoly(ADP-ribose) Polymerase, on Cultured Tumor Cells”, Oncol. Res., 6:9,399-403 (1994), discusses the inhibition of PARP activity, reducedproliferation of tumor cells, and a marked synergistic effect when tumorcells are co-treated with an alkylating drug.

A recent comprehensive review of the state of the art has been publishedby Li and Zhang in IDrugs 2001, 4(7): 804-812.

There continues to be a need for effective and potent PARP inhibitors,and more particularly PARP-1 inhibitors which produce minimal sideeffects. The present invention provides compounds, compositions for, andmethods of, inhibiting PARP activity for treating cancer and/orpreventing cellular, tissue and/or organ damage resulting from celldamage or death due to, for example, necrosis or apoptosis. Thecompounds and compositions of the present invention are especiallyuseful in enhancing the effectiveness of chemotherapy and radiotherapywhere a primary effect of the treatment is that of causing DNA damage inthe targeted cells.

BACKGROUND PRIOR ART

EP 371564, published on Jun. 6, 1990, discloses (1H-azol-1-ylmethyl)substituted quinoline, quinazoline or quinoxaline derivatives. Thedescribed compounds suppress the plasma elimination of retinoic acids.More in particular the compounds3-ethyl-6-[2-methyl-1-(1H-1,2,4-triazol-1-yl)propyl]-2(1H)-quinoxalinone(compound No. 20 in the present application),3-ethyl-6-[1-(1H-imidazol-1-yl)-2-methylpropyl]-2(1H)-quinoxalinone(compound No. 21 in the present application),6-[2-methyl-1-(1H-1,2,4-triazol-1-yl)propyl]-3-(2-thienyl)-2(1H)-quinoxalinone(compound No. 22 in the present application),6-[2-methyl-1-(1H-1,2,4-triazol-1-yl)propyl]-3-(thienyl)-2(1H)-quinoxalinone(compound No. 23 in the present application),6-[1-(1H-imidazol-1-yl)-2-methylpropyl]-3-(3-thienyl)-2(1H)-quinoxalinone(compound No. 24 in the present application) and6-[1-(1H-imidazol-1-yl)pentyl]-3-methyl-2(1H)-quinoxalinone (compoundNo. 25 in the present application) are disclosed.

DESCRIPTION OF THE INVENTION

This invention concerns compounds of formula (I)

the N-oxide forms, the addition salts and the stereo-chemically isomericforms thereof, wherein

-   n is 0, 1 or 2;-   X is N or CR⁵, wherein R⁵ is hydrogen or taken together with R¹ may    form a bivalent radical of formula —CH═CH—CH═CH—;-   R¹ is C₁₋₆alkyl or thienyl;-   R² is hydrogen or hydroxy or taken together with R³ or R⁴ may form    ═O;-   R³ is a radical selected from

—(CH₂)_(s)—NR⁶R⁷  (a-1),

—O—H  (a-2),

—O—R⁸  (a-3),

—S—R⁹  (a-4), or

—C≡N  (a-5),

-   -   wherein    -   s is 0, 1, 2 or 3;    -   R⁶ is —CHO, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, C₁₋₆alkylcarbonyl,        di(C₁₋₆alkyl)aminoC₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl,        C₁₋₆alkylcarbonylaminoC₁₋₆alkyl,        piperidinylC₁₋₆alkylaminocarbonyl, piperidinyl,        piperidinylC₁₋₆alkyl, piperidinylC₁₋₆alkylaminocarbonyl,        C₁₋₆alkyloxy, thienylC₁₋₆alkyl, pyrrolylC₁₋₆alkyl,        arylC₁₋₆alkylpiperidinyl, arylcarbonylC₁₋₆alkyl,        arylcarbonylpiperidinylC₁₋₆alkyl,        haloindozolylpiperidinylC₁₋₆alkyl, or        arylC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl;    -   R⁷ is hydrogen or C₁₋₆alkyl;    -   R⁸ is C₁₋₆alkyl, C₁₋₆alkylcarbonyl or        di(C₁₋₆alkyl)aminoC₁₋₆alkyl; and    -   R⁹ is di(C₁₋₆alkyl)aminoC₁₋₆alkyl;        or R³ is a group of formula

—Z—  (b-1),

-   -   wherein    -   Z is a heterocyclic ring system selected from

-   -   wherein each R¹⁰ independently is hydrogen, C₁₋₆alkyl,        aminocarbonyl, hydroxy,

-   -   C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkylamino,        arylC₁₋₆alkyl, di(phenylC₂₋₆alkenyl), piperidinylC₁₋₆alkyl,        C₃₋₁₀cycloalkyl, C₃₋₁₀cycloalkylC₁₋₆alkyl,        aryloxy(hydroxy)C₁₋₆alkyl, haloindazolyl, arylC₁₋₆alkyl,        arylC₂₋₆alkenyl, morpholino, C₁₋₆alkylimidazolyl, or        pyridinylC₁₋₆alkylamino;    -   R⁴ is hydrogen, C₁₋₆alkyl, furanyl, pyridinyl, arylC₁₋₆alkyl or

-   -   aryl is phenyl or phenyl substituted with halo, C₁₋₆alkyl or        C₁₋₆alkyloxy;    -   with the proviso that when    -   n is 0, X is N, R² is hydrogen, R³ is a group of formula (b-1),        Z is the heterocyclic ring system (c-2) or (c-4) wherein said        heterocyclic ring system Z is attached to the rest of the        molecule with a nitrogen atom, and R¹⁰ is hydrogen; then    -   R⁴ is other than C₁₋₆alkyl or pyridinyl.

Whenever the heterocyclic ring system Z contains a —CH₂—, —CH═, or —NH—moiety the substituent R¹⁰ or the rest of the molecule can be attachedto the carbon or nitrogen atom in which case one or both hydrogen atomsare replaced.

The compounds of formula (I) may also exist in their tautomeric forms.Such forms although not explicitly indicated in the above formula areintended to be included within the scope of the present invention.

A number of terms used in the foregoing definitions and hereinafter areexplained hereunder. These terms are sometimes used as such or incomposite terms.

As used in the foregoing definitions and hereinafter, halo is generic tofluoro, chloro, bromo and iodo; C₁₋₆alkyl defines straight and branchedchain saturated hydrocarbon radicals having from 1 to 6 carbon atomssuch as, e.g. methyl, ethyl, propyl, butyl, pentyl, hexyl,1-methylethyl, 2-methylpropyl, 2-methyl-butyl, 2-methylpentyl and thelike; C₁₋₆alkanediyl defines bivalent straight and branched chainedsaturated hydrocarbon radicals having from 1 to 6 carbon atoms such as,for example, methylene, 1,2-ethanediyl, 1,3-propanediyl-1,4-butanediyl,1,5-pentanediyl, 1,6-hexanediyl and the branched isomers thereof suchas, 2-methylpentanediyl, 3-methylpentanediyl, 2,2-dimethylbutanediyl,2,3-dimethylbutanediyl and the like; C₂₋₆alkenyl defines straight andbranched chain hydrocarbon radicals containing one double bond andhaving from 2 to 6 carbon atoms such as, for example, ethenyl,2-propenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl, andthe like; C₃₋₁₀cycloalkyl includes cyclic hydrocarbon groups having from3 to 10 carbons, such as cyclopropyl, cyclobutyl, cyclopentyl,cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl and thelike.

The term “addition salt” comprises the salts which the compounds offormula (I) are able to form with organic or inorganic bases such asamines, alkali metal bases and earth alkaline metal bases, or quaternaryammonium bases, or with organic or inorganic acids, such as mineralacids, sulfonic acids, carboxylic acids or phosphorus containing acids.

The term “addition salt” further comprises pharmaceutically acceptablesalts, metal complexes and solvates and the salts thereof, that thecompounds of formula (I) are able to form.

The term “pharmaceutically acceptable salts” means pharmaceuticallyacceptable acid or base addition salts. The pharmaceutically acceptableacid or base addition salts as mentioned hereinabove are meant tocomprise the therapeutically active non-toxic acid and non-toxic baseaddition salt forms which the compounds of formula (I) are able to form.The compounds of formula (I) which have basic properties can beconverted in their pharmaceutically acceptable acid addition salts bytreating said base form with an appropriate acid. Appropriate acidscomprise, for example, inorganic acids such as hydrohalic acids, e.g.hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and thelike acids; or organic acids such as, for example, acetic, propanoic,hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e.butanedioic acid), maleic, fumaric, malic, tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.

The compounds of formula (I) which have acidic properties may beconverted in their pharmaceutically acceptable base addition salts bytreating said acid form with a suitable organic or inorganic base.Appropriate base salt forms comprise, for example, the ammonium salts,the alkali and earth alkaline metal salts, e.g. the lithium, sodium,potassium, magnesium, calcium salts and the like, salts with organicbases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, andsalts with amino acids such as, for example, arginine, lysine and thelike.

The terms acid or base addition salt also comprise the hydrates and thesolvent addition forms which the compounds of formula (I) are able toform. Examples of such forms are e.g. hydrates, alcoholates and thelike.

The term “metal complexes” means a complex formed between a compound offormula (I) and one or more organic or inorganic metal salt or salts.Examples of said organic or inorganic salts comprise the halogenides,nitrates, sulfates, phosphates, acetates, trifluoroacetates,trichloroacetates, propionates, tartrates, sulfonates, e.g.methylsulfonates, 4-methylphenylsulfonates, salicylates, benzoates andthe like of the metals of the second main group of the periodicalsystem, e.g. the magnesium or calcium salts, of the third or fourth maingroup, e.g. aluminium, tin, lead as well as the first to the eighthtransition groups of the periodical system such as, for example,chromium, manganese, iron, cobalt, nickel, copper, zinc and the like.

The term stereochemically isomeric forms of compounds of formula (I), asused hereinbefore, defines all possible compounds made up of the sameatoms bonded by the same sequence of bonds but having differentthree-dimensional structures which are not interchangeable, which thecompounds of formula (I) may possess. Unless otherwise mentioned orindicated, the chemical designation of a compound encompasses themixture of all possible stereochemically isomeric forms which saidcompound may possess. Said mixture may contain all diastereomers and/orenantiomers of the basic molecular structure of said compound. Allstereochemically isomeric forms of the compounds of formula (I) both inpure form or in admixture with each other are intended to be embracedwithin the scope of the present invention.

The N-oxide forms of the compounds of formula (I) are meant to comprisethose compounds of formula (I) wherein one or several nitrogen atoms areoxidized to the so-called N-oxide, particularly those N-oxides whereinone or more of the piperidine-, piperazine or pyridazinyl-nitrogens areN-oxidized.

Whenever used hereinafter, the term “compounds of formula (I)” is meantto include also the N-oxide forms, the pharmaceutically acceptable acidor base addition salts and all stereoisomeric forms.

The compounds described in EP 371564 suppress the plasma elimination ofretinoic acids. The compounds3-ethyl-6-[2-methyl-1-(1H-1,2,4-triazol-1-yl)propyl]-2(1H)-quinoxalinone(compound No. 20 in the present application),3-ethyl-6-[1-(1H-imidazol-1-yl)-2-methylpropyl]-2(1H)-quinoxalinone(compound No. 21 in the present application),6-[2-methyl-1-(1H-1,2,4-triazol-1-yl)propyl]-3-(2-thienyl)-2(1H)-quinoxalinone(compound No. 22 in the present application),6-[2-methyl-1-(1H-1,2,4-triazol-1-yl)propyl]-3-(thienyl)-2(1H)-quinoxalinone(compound No. 23 in the present application),6-[1-(1H-imidazol-1-yl)-2-methylpropyl]-3-(3-thienyl)-2(1H)-quinoxalinone(compound No. 24 in the present application) and6-[1-(1H-imidazol-1-yl)pentyl]-3-methyl-2(1H)-quinoxalinone (compoundNo. 25 in the present application) have been disclosed in EP 371564.Unexpectedly, it has been found that the compounds of the presentinvention show PARP inhibitory activity.

A first group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) n is 0 or 1;-   b) X is N or CR⁵, wherein R⁵ is hydrogen;-   c) R³ is a radical selected from (a-1), (a-2) or (a-3) or is a group    of formula (b-1) i.e. —Z—;-   d) s is 0, 1 or 2;-   e) R⁶ is —CHO, C₁₋₆alkyl, piperidinylC₁₋₆alkyl,    arylcarbonylpiperidinylC₁₋₆alkyl or    arylC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl;-   f) R⁸ is C₁₋₆alkyl;-   g) when R³ is a group of formula (b-1) then Z is a heterocyclic ring    system selected from (c-2) or (c-4); and-   h) each R¹⁰ independently is hydrogen, C₁₋₆alkyl or    C₁₋₆alkyloxyC₁₋₆alkylamino.

A second group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

a) n is 0;b) X is N or CR⁵, wherein R⁵ is hydrogen;c) R¹ is C₁₋₆alkyl;d) R² is hydrogen or hydroxy or taken together with R⁴ may form ═O;e) R³ is a radical selected from (a-1) or (a-2);f) s is 0 or 1;g) R⁶ is —CHO or C₁₋₆alkyl; and

h) R⁴ is hydrogen, C₁₋₆alkyl or

-   A third group of interesting compounds consists of those compounds    of formula (I), the first group of interesting compounds or the    second group of interesting compounds wherein Z is a heterocyclic    ring system other than the heterocyclic ring system of formula (c-2)    or (c-4).-   A group of preferred compounds consists of those compounds of    formula (I) wherein n is 0 or 1; X is N or CR⁵, wherein R⁵ is    hydrogen; R³ is a radical selected from (a-1), (a-2) or (a-3) or is    a group of formula (b-1) i.e. —Z—; s is 0, 1 or 2; R⁶ is —CHO,    C₁₋₆alkyl, piperidinylC₁₋₆alkyl, arylcarbonylpiperidinylC₁₋₆alkyl or    arylC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl; R⁸ is C₁₋₆alkyl; when R³ is    a group of formula (b-1) then Z is a heterocyclic ring system    selected from (c-2) or (c-4); and each R¹⁰ independently is    hydrogen, C₁₋₆alkyl or C₁₋₆alkyloxyC₁₋₆alkylamino.-   A further group of preferred compounds consists of those compounds    of formula (I) wherein n is 0; X is N or CR⁵, wherein R⁵ is    hydrogen; R¹ is C₁₋₆alkyl;    R² is hydrogen or hydroxy or taken together with R⁴ may form ═O; R³    is a radical selected from (a-1) or (a-2); s is 0 or 1; R⁶ is —CHO    or C₁₋₆alkyl; and R⁴ is hydrogen, C₁₋₆alkyl or

-   An even further group of preferred compounds consists of those    compounds of formula (I), the group of preferred compounds or the    further group of preferred compounds wherein Z is a heterocyclic    ring system other than the heterocyclic ring system of formula (c-2)    or (c-4).

The most preferred compounds are compound No 1, compound No 5, compoundNo 7, compound No 3 and compound No 17.

The compounds of formula (I) can be prepared according to the generalmethods described in EP 371564.

A number of such preparation methods will be described hereinafter inmore detail.

Other methods for obtaining final compounds of formula (I) are describedin the examples.

Compounds of formula (I) wherein R² is hydrogen and R³ is —NR⁷—CHOwherein R⁷ is hydrogen or methyl, herein referred to as compounds offormula (I-b), can be prepared starting from compounds of formula (I),wherein R² taken together with R³ forms ═O, herein referred to ascompounds of formula (I-a), in the presence of formamide ormethylformamide, here indicated as intermediates of formula (II), andformic acid.

Compounds of formula (I), wherein R³ is hydroxy, herein referred to ascompounds of formula (I-c), can be prepared by converting the ketonmoiety of compounds of formula (I-a) into an hydroxy group, with anappropriate reductant, e.g., sodium borohydride in a suitable solvent,e.g. methanol and tetrahydrofuran.

Compounds of formula (I-a) can be prepared by converting compounds offormula (I-c), wherein R² is hydrogen, herein referred to as compoundsof formula (I-c-1), in the presence of a suitable oxidant such aschromium trioxide and an acid such as sulfuric acid, in a suitablesolvent such as 2-propanone.

Intermediates of formula (IV), wherein W is an appropriate leaving groupsuch as, for example, chloro, bromo, methanesulfonyloxy orbenzenesulfonyloxy can be prepared from compounds of formula (I-c-1) bytreating said compounds with a suitable reagent e.g. methanesulfonyloxychloride or benzenesulfonyloxy chloride, or a halogenating reagent suchas e.g. POCl₃ or SOCl₂.

Compounds of formula (I), defined as compounds of formula (I) whereinR^(b) is as defined in R⁶ and R^(c) is as defined in R⁷, or R^(b) andR^(c) taken together with the nitrogen to which they are attached, forman appropriate heterocyclic ring system as defined in Z, herein referredto as compounds of formula (I-h), can be prepared by reacting anintermediate of formula (IV) with an intermediate of formula (V). Thereaction can be performed in a reaction-inert solvent such asdimethylformamide or acetonitrile, and optionally in the presence of asuitable base such as, for example, sodium carbonate, potassiumcarbonate or thriethylamine.

The compounds of formula (I) may also be converted into each other viaart-known reactions or functional group transformations. A number ofsuch transformations are already described hereinabove. Other examplesare hydrolysis of carboxylic esters to the corresponding carboxylic acidor alcohol; hydrolysis of amides to the corresponding carboxylic acidsor amines; hydrolysis of nitriles to the corresponding amides; aminogroups on imidazole or phenyl may be replaced by a hydrogen by art-knowndiazotation reactions and subsequent replacement of the diazo-group byhydrogen; alcohols may be converted into esters and ethers; primaryamines may be converted into secondary or tertiary amines; double bondsmay be hydrogenated to the corresponding single bond; an iodo radical ona phenyl group may be converted in to an ester group by carbon monoxideinsertion in the presence of a suitable palladium catalyst.

Hence, compounds of formula (I), (I-a), (I-b), (I-c), (I-c-1), (I-h),(I-i), (I-j) and (I-k) can optionally be the subject of one or more ofthe following conversions in any desired order:

(i) converting a compound of formula (I) into a different compound offormula (I);(ii) converting a compound of formula (I) into the correspondingacceptable salt or N-oxide thereof;(iii) converting a pharmaceutically acceptable salt or N-oxide of acompound of formula (I) into the parent compound of formula (I);(iv) preparing a stereochemical isomeric form of a compound of formula(I) or a pharmaceutically acceptable salt or N-oxide thereof.

Intermediates of formula (VII), wherein R^(d) and R^(e) are appropriateradicals or taken together with the carbon to which they are attached,form an appropriate heterocyclic ring system as defined in Z, can beprepared by hydrolysing intermediates of formula (VI), wherein R³ is agroup of formula (b-1) or a radical of formula (a-1) wherein s is otherthan 0, herein referred to as R^(g), according to art-known methods,such as stirring the intermediate (VI) in an aqueous acid solution inthe presence of a reaction inert solvent, e.g. tetrahydrofuran. Anappropriate acid is for instance hydrochloric acid.

Compounds of formula (I) wherein R² is hydrogen and R^(g) is as definedabove, herein referred to as compounds of formula (I-k), can be preparedstarting from intermediates of formula (VII), by a selectivehydrogenation of said intermediate with an appropriate reducing agentsuch as, for example with a noble catalyst, such asplatinum-on-charcoal, palladium-on-charcoal and the like and anappropriate reductant such as hydrogen in a suitable solvent such asmethanol.

Compounds of formula (I) can be prepared by hydrolysing intermediates offormula (VIII), according to art-known methods, by submitting theintermediates of formula (VIII) to appropriate reagents, such as,tinchloride, acetic acid and hydrochloric acid, in the presence of areaction inert solvent, e.g. tetrahydrofuran.

Compounds of formula (I) can be prepared starting from N-oxides offormula (IX) by converting the intermediates of formula (IX) in thepresence of a suitable reagent such as sodium carbonate or aceticanhydride and when appropriate in a solvent such as dichloromethane.

The compounds of formula (I) wherein X is CH herein referred to ascompounds of formula (I-j), may also be obtained by cyclizing anintermediate of formula (X).The cyclization reaction of intermediates offormula (X) may be conducted according to art-known cyclizingprocedures. Preferably the reaction is carried out in the presence of asuitable Lewis Acid, e.g. aluminum chloride either neat or in a suitablesolvent such as, for example, an aromatic hydrocarbon, e.g. benzene,chlorobenzene, methylbenzene and the like; halogenated hydrocarbons,e.g. trichloromethane, tetrachloromethane and the like; an ether, e.g.tetrahydrofuran, 1,4-dioxane and the like; or mixtures of such solvents.Somewhat elevated temperatures, preferably between 70°-100° C., andstirring may enhance the rate of the reaction.

The compounds of formula (I), wherein X is N, herein referred to ascompounds of formula (I-i) may be obtained by condensing an appropriateortho-benzenediamine of formula (XI) with an ester of formula (XII)wherein R^(h) is C₁₋₆alkyl. The condensation of the substitutedortho-diamine of formula (XI) and the ester of formula (XII) can becarried out in the presence of a carboxylic acid, e.g. acetic acid andthe like, a mineral acid such as, for example hydrochloric acid,sulfuric acid, or a sulfonic acid such as, for example, methanesulfonicacid, benzenesulfonic acid, 4-methylbenzenesulfonic acid and the like.Somewhat elevated temperatures may be appropriate to enhance the rate ofthe reaction and in some cases the reaction may even be carried out atthe reflux temperature of the reaction mixture. The water which isliberated during the condensation may be removed from the mixture byazeotropical distillation, distillation and the like methods.

Intermediates of formula (XI) can be prepared by a nitro to aminereduction reaction starting with an intermediate of formula (XIII) inthe presence of a metal catalyst such as Raney Nickel and an appropriatereductant such as hydrogen in a suitable solvent such as methanol.

Intermediates of formula (XIII) can be prepared by hydrolysingintermediates of formula (XIV), according to art-known methods, such asstirring the intermediate (XIV) in an aqueous acid solution in thepresence of a reaction inert solvent, e.g. tetrahydrofuran. Anappropriate acid is for instance hydrochloric acid.

Intermediates of formula (X) can conveniently be prepared by reacting ananiline of formula (XV) with a halide of formula (XVI) in the presenceof a base such as pyridine in a suitable solvent such asdichloromethane.

Intermediates of formula (VIII) wherein n is 0, R² is hydrogen orhydroxy and when R² is hydrogen then R³ is hydroxy herein referred to asintermediates of formula (VIII-a) can be prepared by treating anintermediate of formula (XVII), wherein W is halo, with an organolithiumreagent such as, e.g. n-butyllithium in a reaction inert solvent, e.g.tetrahydrofuran, and subsequently reacting said intermediate with anintermediate of formula (XVIII) wherein R^(i) is hydrogen or a radicalas defined in R³.

The present invention also relates to a compound of formula (I) asdefined above for use as a medicine.

The compounds of the present invention have PARP inhibiting propertiesas can be seen from the experimental part hereinunder.

The present invention also contemplates the use of compounds in thepreparation of a medicament for the treatment of any of the diseases anddisorders in an animal described herein, wherein said compounds arecompounds of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein

-   n is 0, 1 or 2;-   X is N or CR⁵, wherein R⁵ is hydrogen or taken together with R¹ may    form a bivalent radical of formula —CH═CH—CH═CH—;-   R¹ is C₁₋₆alkyl or thienyl;-   R² is hydrogen or hydroxy or taken together with R³ or R⁴ may form    ═O;-   R³ is a radical selected from

—(CH₂)_(s)—NR⁶R⁷  (a-1),

—O—H  (a-2),

—O—R⁸  (a-3),

—S—R⁹  (a-4), or

—C≡N  (a-5),

-   -   wherein    -   s is 0, 1, 2 or 3;    -   R⁶ is —CHO, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, C₁₋₆alkylcarbonyl,        di(C₁₋₆alkyl)aminoC₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkyl,        C₁₋₆alkylcarbonylaminoC₁₋₆alkyl,        piperidinylC₁₋₆alkylaminocarbonyl, piperidinyl,        piperidinylC₁₋₆alkyl, piperidinylC₁₋₆alkylaminocarbonyl,        C₁₋₆alkyloxy, thienylC₁₋₆alkyl, pyrrolylC₁₋₆alkyl,        arylC₁₋₆alkylpiperidinyl, arylcarbonylC₁₋₆alkyl,        arylcarbonylpiperidinylC₁₋₆alkyl,        haloindozolylpiperidinylC₁₋₆alkyl, or        arylC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl;    -   R⁷ is hydrogen or C₁₋₆alkyl;    -   R⁸ is C₁₋₆alkyl, C₁₋₆alkylcarbonyl or        di(C₁₋₆alkyl)aminoC₁₋₆alkyl; and    -   R⁹ is di(C₁₋₆alkyl)aminoC₁₋₆alkyl;        or R³ is a group of formula

—Z—  (b-1),

-   -   wherein    -   Z is a heterocyclic ring system selected from

-   -   wherein each R¹⁰ independently is hydrogen, C₁₋₆alkyl,        aminocarbonyl, hydroxy,

-   -   C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkylamino,        arylC₁₋₆alkyl, di(phenylC₂₋₆alkenyl), piperidinylC₁₋₆alkyl,        C₃₋₁₀cycloalkyl, C₃₋₁₀cycloalkylC₁₋₆alkyl,        aryloxy(hydroxy)C₁₋₆alkyl, haloindazolyl, arylC₁₋₆alkyl,        arylC₂₋₆alkenyl, morpholino, C₁₋₆alkylimidazolyl, or        pyridinylC₁₋₆alkylamino;        R⁴ is hydrogen, C₁₋₆alkyl, furanyl, pyridinyl, C₁₋₆alkylaryl or

aryl is phenyl or phenyl substituted with halo, C₁₋₆alkyl orC₁₋₆alkyloxy.

In view of their PARP binding properties the compounds of the presentinvention may be used as reference compounds or tracer compounds inwhich case one of the atoms of the molecule may be replaced with, forinstance, a radioactive isotope.

To prepare the pharmaceutical compositions of this invention, aneffective amount of a particular compound, in base or acid addition saltform, as the active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirably inunitary dosage form suitable, preferably, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules and tablets. Because of their ease in administration, tabletsand capsules represent the most advantageous oral dosage unit form, inwhich case solid pharmaceutical carriers are obviously employed. Forparenteral compositions, the carrier will usually comprise sterilewater, at least in large part, though other ingredients, to aidsolubility for example, may be included. Injectable solutions, forexample, may be prepared in which the carrier comprises saline solution,glucose solution or a mixture of saline and glucose solution. Injectablesuspensions may also be prepared in which case appropriate liquidcarriers, suspending agents and the like may be employed. In thecompositions suitable for percutaneous administration, the carrieroptionally comprises a penetration enhancing agent and/or a suitablewetting agent, optionally combined with suitable additives of any naturein minor proportions, which additives do not cause a significantdeleterious effect to the skin. Said additives may facilitate theadministration to the skin and/or may be helpful for preparing thedesired compositions. These compositions may be administered in variousways, e.g., as a transdermal patch, as a spot-on, as an ointment. It isespecially advantageous to formulate the aforementioned pharmaceuticalcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used in the specification andclaims herein refers to physically discrete units suitable as unitarydosages, each unit containing a predetermined quantity of activeingredient calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Examples of suchdosage unit forms are tablets (including scored or coated tablets),capsules, pills, powder packets, wafers, injectable solutions orsuspensions, teaspoonfuls, tablespoonfuls and the like, and segregatedmultiples thereof.

The compounds of the present invention can treat or prevent tissuedamage resulting from cell damage or death due to necrosis or apoptosis;can ameliorate neural or cardiovascular tissue damage, including thatfollowing focal ischemia, myocardial infarction, and reperfusion injury;can treat various diseases and conditions caused or exacerbated by PARPactivity; can extend or increase the lifespan or proliferative capacityof cells; can alter the gene expression of senescent cells; canradiosensitize and/or chemosensitize cells. Generally, inhibition ofPARP activity spares the cells from energy loss, preventing, in the caseof neural cells, irreversible depolarization of the neurons, and thus,provides neuroprotection.

For the foregoing reasons, the present invention further relates to amethod of administering a therapeutically effective amount of theabove-identified compounds in an amount sufficient to inhibit PARPactivity, to treat or prevent tissue damage resulting from cell damageor death due to necrosis or apoptosis, to effect a neuronal activity notmediated by NMDA toxicity, to effect a neuronal activity mediated byNMDA toxicity, to treat neural tissue damage resulting from ischemia andreperfusion injury, neurological disorders and neurodegenerativediseases; to prevent or treat vascular stroke; to treat or preventcardiovascular disorders; to treat other conditions and/or disorderssuch as age-related muscular degeneration, AIDS and other immunesenescence diseases, inflammation, gout, arthritis, atherosclerosis,cachexia, cancer, degenerative diseases of skeletal muscle involvingreplicative senescence, diabetes, head trauma, inflammatory boweldisorders (such as colitis and Crohn's disease), muscular dystrophy,osteoarthritis, osteoporosis, chronic and/or acute pain (such asneuropathic pain), renal failure, retinal ischemia, septic shock (suchas endotoxic shock), and skin aging, to extend the lifespan andproliferative capacity of cells; to alter gene expression of senescentcells; chemosensitize and/or radiosensitize (hypoxic) tumor cells. Thepresent invention also relates to treating diseases and conditions in ananimal which comprises administering to said animal a therapeuticallyeffective amount of the above-identified compounds.

In particular, the present invention relates to a method of treating,preventing or inhibiting a neurological disorder in an animal, whichcomprises administering to said animal a therapeutically effectiveamount of the above-identified compounds. The neurological disorder isselected from the group consisting of peripheral neuropathy caused byphysical injury or disease state, traumatic brain injury, physicaldamage to the spinal cord, stroke associated with brain damage, focalischemia, global ischemia, reperfusion injury, demyelinating disease andneurological disorder relating to neurodegeneration.

The present invention also contemplates the use of compounds of formula(I) for inhibiting PARP activity, for treating, preventing or inhibitingtissue damage resulting from cell damage or death due to necrosis orapoptosis, for treating, preventing or inhibiting a neurologicaldisorder in an animal.

The term “preventing neurodegeneration” includes the ability to preventneurodegeneration in patients newly diagnosed as having aneurodegenerative disease, or at risk of developing a new degenerativedisease and for preventing further neurodegeneration in patients who arealready suffering from or have symptoms of a neurodegenerative disease.

The term “treatment” as used herein covers any treatment of a diseaseand/or condition in an animal, particularly a human, and includes: (i)preventing a disease and/or condition from occurring in a subject whichmay be predisposed to the disease and/or condition but has not yet beendiagnosed as having it; (ii) inhibiting the disease and/or condition,i.e., arresting its development; (iii) relieving the disease and/orcondition, i.e., causing regression of the disease and/or condition.

The term “radiosensitizer”, as used herein, is defined as a molecule,preferably a low molecular weight molecule, administered to animals intherapeutically effective amounts to increase the sensitivity of thecells to ionizing radiation and/or to promote the treatment of diseaseswhich are treatable with ionizing radiation. Diseases which aretreatable with ionizing radiation include neoplastic diseases, benignand malignant tumors, and cancerous cells. Ionizing radiation treatmentof other diseases not listed herein are also contemplated by the presentinvention.

The term “chemosensitizer”, as used herein, is defined as a molecule,preferably a low molecular weight molecule, administered to animals intherapeutically effective amounts to increase the sensitivity of cellsto chemotherapy and/or promote the treatment of diseases which aretreatable with chemotherapeutics. Diseases which are treatable withchemotherapy include neoplastic diseases, benign and malignant tumorsand cancerous cells. Chemotherapy treatment of other diseases not listedherein are also contemplated by the present invention.

The compounds, compositions and methods of the present invention areparticularly useful for treating or preventing tissue damage resultingfrom cell death or damage due to necrosis or apoptosis.

The compounds of the present invention can be “anti-cancer agents”,which term also encompasses “anti-tumor cell growth agents” and“anti-neoplastic agents”. For example, the methods of the invention areuseful for treating cancers and chemosensitizing and/or radiosensitizingtumor cells in cancers such as ACTH-producing tumors, acute lymphocyticleukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex,bladder cancer, brain cancer, breast cancer, cervical cancer, chroniclymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer,cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer,Ewing's sarcoma gallbladder cancer, hairy cell leukemia, head & neckcancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, livercancer, lung cancer (small and/or non-small cell), malignant peritonealeffusion, malignant pleural effusion, melanoma, mesothelioma, multiplemyeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovariancancer, ovary (germ cell) cancer, prostate cancer, pancreatic cancer,penile cancer, retinoblastoma, skin cancer, soft tissue sarcoma,squamous cell carcinomas, stomach cancer, testicular cancer, thyroidcancer, trophoblastic neoplasms, uterine cancer, vaginal cancer, cancerof the vulva and Wilm's tumor.

Hence the compounds of the present invention can be used as“radiosensitizer” and/or “chemosensitizer”.

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of ionizing radiation. Several mechanisms forthe mode of action of radiosensitizers have been suggested in theliterature including: hypoxic cell radiosensitizers (e.g.,2-nitroimidazole compounds, and benzotriazine dioxide compounds)mimicking oxygen or alternatively behave like bioreductive agents underhypoxia; non-hypoxic cell radiosensitizers (e.g., halogenatedpyrimidines) can be analogs of DNA bases and preferentially incorporateinto the DNA of cancer cells and thereby promote the radiation-inducedbreaking of DNA molecules and/or prevent the normal DNA repairmechanisms; and various other potential mechanisms of action have beenhypothesized for radiosensitizers in the treatment of disease. Manycancer treatment protocols currently employ radiosensitizers inconjunction with radiation of x-rays. Examples of x-ray activatedradiosensitizers include, but are not limited to, the following:metronidazole, misonidazole, desmethylmisonidazole, pimonidazole,etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145,nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin,and therapeutically effective analogs and derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives, tinetioporphyrin, pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines,phthalocyanines, zinc phthalocyanine, and therapeutically effectiveanalogs and derivatives of the same.

Radiosensitizers may be administered in conjunction with atherapeutically effective amount of one or more other compounds,including but not limited to: compounds which promote the incorporationof radiosensitizers to the target cells; compounds which control theflow of therapeutics, nutrients, and/or oxygen to the target cells;chemotherapeutic agents which act on the tumor with or withoutadditional radiation; or other therapeutically effective compounds fortreating cancer or other disease. Examples of additional therapeuticagents that may be used in conjunction with radiosensitizers include,but are not limited to: 5-fluorouracil, leucovorin, 5′-amino-5′deoxythymidine, oxygen, carbogen, red cell transfusions,perfluorocarbons (e.g., Fluosol 10 DA), 2,3-DPG, BW12C, calcium channelblockers, pentoxyfylline, antiangiogenesis compounds, hydralazine, andLBSO. Examples of chemotherapeutic agents that may be used inconjunction with radiosensitizers include, but are not limited to:adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2,irinotecan, paclitaxel, topotecan, and therapeutically effective analogsand derivatives of the same.

Chemosensitizers may be administered in conjunction with atherapeutically effective amount of one or more other compounds,including but not limited to: compounds which promote the incorporationof chemosensitizers to the target cells; compounds which control theflow of therapeutics, nutrients, and/or oxygen to the target cells;chemotherapeutic agents which act on the tumor or other therapeuticallyeffective compounds for treating cancer or other disease. Examples ofadditional therapeutica agents that may be used in conjunction withchemosensitizers include, but are not limited to: methylating agents,toposisomerase I inhibitors and other chemotherapeutic agents such ascisplatin and bleomycin.

The compounds of formula (I) can also be used to detect or identify thePARP, and more in particular the PARP-1 receptor. For that purpose thecompounds of formula (I) can be labeled. Said label can be selected fromthe group consisting of a radioisotope, spin label, antigen label,enzyme label fluorescent group or a chemiluminiscent group.

Those skilled in the art could easily determine the effective amountfrom the test results presented hereinafter. In general it iscontemplated that an effective amount would be from 0.01 mg/kg to 100mg/kg body weight, and in particular from 0.05 mg/kg to 10 mg/kg bodyweight. It may be appropriate to administer the required dose as two,three, four or more sub-doses at appropriate intervals throughout theday. Said sub-doses may be formulated as unit dosage forms, for example,containing 0.5 to 500 mg, and in particular 1 mg to 200 mg of activeingredient per unit dosage form.

The following examples illustrate the present invention.

EXPERIMENTAL PART

Hereinafter, “BuLi” is defines as butyl-lithium, “DCM” is defined asdichloromethane, “DIPE” is defined as diisopropyl ether, “DMF” isdefined as N,N-dimethylformamide, “EtOAc” is defined as ethyl acetate,“EtOH” is defined as ethanol, “MEK” is defined as methyl ethyl keton,“MeOH” is defined as methanol and “THF” is defined as tetrahydrofuran.

A. Preparation of the Intermediate Compounds Example A1 a) Preparationof Intermediate 1

A mixture of 1-(4-amino-3-nitrophenyl)-2-methyl-1-propanone (0.0144 mol)in formic acid (4.93 ml) and formamide (18.2 ml) was stirred at 160° C.for 15 hours, then cooled to room temperature, poured out into icewater, basified with a concentrated ammonium hydroxidesolution andextracted with EtOAc. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated till dryness, yielding 4.8 g ofintermediate 1.

b) Preparation of Intermediate 2

A mixture of intermediate 1 (0.0144 mol) in MeOH (50 ml) washydrogenated under a 3 bar pressure for 1 hour with Raney Nickel (3.4 g)as a catalyst. After uptake of H₂ (3 equiv), the catalyst was filteredthrough celite, washed with MeOH and the filtrate was evaporated tilldryness. The product was used without further purification, yielding 4.7g of intermediate 2.

Example A2 Preparation of Intermediate 3 and 4

Aluminium chloride (0.6928 mol) was added portionwise to a solution ofchloro-acetyl chloride (0.5196 mol) in DCM (50.2 ml) while thetemperature was kept below 30° C. 3-ethyl-2(1H)-quinolinone (0.1732 mol)was added while the temperature was kept below 30° C. The mixture wasstirred and refluxed for 15 hours, cooled and poured out into ice water.The precipitate was filtered off, washed with water and taken up in DCM.The organic solution was stirred and filtered. The precipitate wasdried, yielding 33.5 g of intermediate 3. The filtrate was extracted.The organic layer was separated, dried (MgSO₄), filtered and the solventwas evaporated till dryness, yielding 20.46 g of intermediate 4.

Example A3 a) Preparation of Intermediate 5

A mixture of 6-bromo-2-chloro-3-methyl-quinoline (0.04483 mol) andCH₃ONa (0.224 mol) in MeOH (200 ml) was stirred at 70° C. for 36 hours.The mixture was cooled, poured into ice, EtOAc was added and the mixturewas extracted with EtOAc. The organic layer was washed with water, dried(MgSO₄), filtered off and evaporated, yielding 11g (97%) of intermediate5.

b) Preparation of Intermediate 6

BuLi 1.6M in hexane (0.0619 mol) was added dropwise at −60° C. under N₂flow to a mixture of intermediate 5 (0.0476 mol) in THF (200 ml). Themixture was stirred at −60° C. for 1 hour. A mixture of3-(dimethylamino)-1-(2-furanyl)-1-propanone (0.0571 mol) in THF (100 ml)was added dropwise at −60° C. The mixture was stirred at −60° C. for 2hours and then at −40° C. for 1 hour. The mixture was poured out into asaturated ammonium chloride solution and extracted with EtOAc. Theorganic layer was separated, dried (MgSO₄), filtered and the solvent wasevaporated. The product was used without further purification, yielding16.2 g of intermediate 6.

c) Preparation of Intermediate 7

A mixture of intermediate 6 (0.0476 mol) in hydrochloric acid 3N (254ml) and THF (128 ml) was stirred and refluxed for 6 hours. The mixturewas poured out on ice, basified with a concentrated ammonium hydroxidesolution and extracted with EtOAc. The organic layer was separated,dried (MgSO₄), filtered and the solvent was evaporated. The residue waspurified by column chromatography over silica gel (15-40 μm) (eluent:DCM/MeOH/NH₄OH 95/5/0.2). The pure fractions were collected and thesolvent was evaporated, yielding 4g (27%) of intermediate 7.

Example A4 Preparation of Intermediate 8

nBuLi 1.6M in hexane (0.129 mol) was added dropwise at −60° C. under N₂flow to a mixture of 6-bromo-3-ethyl-2-methoxy-quinoline (0.0996 mol) inTHF (265 ml). The mixture was stirred at −60° C. for 1 hour. A mixtureof 2-ethyl-butanal (0.119 mol) in THF (100 ml) was added dropwise at−60° C. The mixture was stirred at −60° C. for 2 hours, then at −40° C.for 1 hour, poured out into a saturated ammonium chloride solution andextracted with EtOAc. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated. The product was used withoutfurther purification, yielding 28.62 g of intermediate 8.

Example A5 a) Preparation of Intermediate 9

A solution of (2-bromoethyl)-benzene (0.174 mol) in diethyl ether (125ml) was added dropwise at 0° C. to a suspension of Mg turnings (0.21mol) in diethyl ether (125 ml) and the mixture was stirred at 0° C. for1 hour. A solution of 3-methyl-6-quinolinecarboxaldehyde (0.116 mol) inTHF (200 ml) was added dropwise at 0° C. and the mixture was stirred atroom temperature for 2 h. The mixture was poured into ice water,filtered through celite and the product was extracted with EtOAc. Theorganic layer was washed with water, dried (MgSO₄), filtered off andevaporated. The residue was crystallized from EtOAc/diethyl ether,yielding 19 g (59%) of intermediate 9.

b) Preparation of Intermediate 10

Potassium permanganate (19g) was added dropwise at 5° C. under N₂ to asolution of intermediate 9 (0.069 mol) in DCM (300 ml) andtris[2-(2-methoxyethoxy)ethyl]amine (2 ml) and the mixture was stirredat room temperature for the night. The mixture was filtered throughcelite and the filtrate was evaporated, yielding 17g (90%) ofintermediate 10.

c) Preparation of Intermediate 11

A solution of 3-chloro-benzenecarboperoxoic acid (0.123 mol) in DCM (200ml) was added at 5° C. under N₂ to a solution of intermediate 10 (0.062mol) in DCM (200 ml), the mixture was stirred at 5° C. for 1 hour andthen at room temperature for 3 hours. Aqueous potassium carbonate 10%was added and the product was extracted with DCM. The organic layer waswashed with water, dried (MgSO₄), filtered off and evaporated. Theproduct was used without further purification, yielding 18g (100%) ofintermediate 11.

d) Preparation of Intermediate 12

Potassium carbonate 10% (250 ml) was added at room temperature to asolution of intermediate 11 (0.062 mol) in DCM (250 ml) and the mixturewas stirred for 10 min. Tosyl chloride (0.093 mol) was added portionwiseand the mixture was stirred at room temperature for 2 hours. Theprecipitate was filtered off, washed with water and dried. The residue(10.1 g) was recrystallized from 2-propanone, yielding 2.8 g (72%) ofintermediate 12.

B. Preparation of the Final Compounds Example B1 Preparation of FinalCompound 1

A mixture of intermediate 2 (0.011 mol) and ethyl 2-oxobutanoate (0.022mol) in EtOH (40 ml) was stirred at 60° C. for 6 hours and then cooledto room temperature. The solvent was evaporated. The residue was takenup in a saturated NaHCO₃ solution. The mixture was extracted with DCM.The organic layer was separated, dried (MgSO₄), filtered and the solventwas evaporated till dryness. The residue was purified by columnchromatography over silica gel (15-40 μm) (eluent: DCM/MeOH/NH₄OH99/1/0.1 and 85/15/0.1). The pure fractions were collected and thesolvent was evaporated. The residue (1.9 g) was purified again by columnchromatography over silica gel (15-40 μm) (eluent:cyclohexane/2-propanol/NH₄OH 88/12/1). The pure fractions were collectedand the solvent was evaporated. The residue was crystallized from DIPE.The precipitate was filtered off and dried, yielding 0.33 g (11%) ofcompound 1, melting point 204° C.

Example B2 Preparation of Final Compound 2

Aluminium chloride (0.234 mol) was added portionwise to a solution ofN-[4-[1-(1H-imidazol-1-yl)-2-methylpropyl]phenyl]-2-methyl-3-phenyl-2-propenamide(0.026 mol) in chloro-benzene (60 ml) and the mixture was stirred at100° C. for 3 hours. The mixture was poured into ice water, basifiedwith NH₄OH and extracted with DCM. The mixture was filtered throughcelite and the filtrate was decanted. The organic layer was dried(MgSO₄), filtered off and evaporated till dryness. The residue waspurified by column chromatography over silica gel (35-70 μm) (eluent:DCM/MeOH/NH₄OH 95/5/0.1). The pure fractions were collected andevaporated. The residue (4 g) was crystallized from MEK, yielding: 2.12g (29%) of compound 2, melting point 211.4° C.

Example B3 Preparation of Final Compound 3

Dimethylamine, hydrochloride (0.3 mol) was added portionwise at roomtemperature under N₂ flow to a suspension of potassium carbonate (0.3603mol) in DMF (300 ml). The mixture was stirred for 30 min. A mixture ofintermediate 3 (0.06 mol) and intermediate 4 (0.06 mol) was addedcarefully. The mixture was stirred at room temperature for 30 min. Icewater was added. The precipitate was filtered off, washed with water andthe filtrate was extracted with DCM. The organic layer was separated,dried (MgSO₄), filtered and the solvent was evaporated till dryness. Theresidue (16.6 g) was purified by column chromatography over silica gel(20-45 μm) (eluent: DCM/MeOH/NH₄OH 95/5/0.2). The pure fractions werecollected and the solvent was evaporated. The residue (4.9 g) wascrystallized from 2-propanone and MeOH. The precipitate was filtered offand dried, yielding 1.2 g of compound 3, melting point 180° C.

Example B4 Preparation of Final Compound 4

A mixture of intermediate 7 (0.0113 mol) in MeOH (60 ml) washydrogenated at 40° C. under a 4.8 bar pressure for 6 h with Pd/C 10%(0.35 g) as a catalyst. After uptake of H₂ (1 eq), the catalyst wasfiltered over celite and the filtrate was evaporated. The residue wastaken up in water and a concentrated ammonium hydroxide solution andextracted with DCM. The organic layer was separated, dried (MgSO₄),filtered and the solvent was evaporated. The residue was purified bycolumn chromatography over silica gel (15-40 μm) (eluent: DCM/MeOH/NH₄OH95/5/0.3 and 93/7/0.5). The pure fractions were collected and thesolvent was evaporated. The residue was crystallized from 2-propanoneand diethyl ether. The precipitate was filtered off and dried, yielding0.69 g (20%) of compound 4.

Example B5 Preparation of Final Compound 5

A mixture of intermediate 8 (0.0996 mol) in hydrochloric acid 3N (426ml) and THF (274 ml) was stirred at 70° C. overnight, then poured out onice, basified with a concentrated NH₄OH solution and extracted withEtOAc. The organic layer was separated, dried (MgSO₄), filtered and thesolvent was evaporated. The residue was crystallized from DCM. Theprecipitate was filtered off and dried, yielding: 15.21 g (56%) ofcompound 5.

Example B6 Preparation of Final Compound 6

A mixture of intermediate 12 (0.013 mol) in formamide (61.8 ml) andformic acid (30 ml) was stirred and refluxed for 36 h. The mixture wascooled to room temperature, poured into ice water and filtered off. Theprecipitate was washed with water, 2-propanone and diethyl ether. Theprecipitate was dried and recrystallized from MeOH/THF, yielding 1.74 g(40%) of compound 6, melting point 221.3° C.

Example B7 Preparation of Final Compound 7

Sodium hydroborate (0.0151 mol) was added at 0° C. under N₂ flow to asolution of compound 3 (0.0116 mol) in MeOH (50 ml). The mixture wasstirred for 1 hour and poured out into water. The organic solvent wasevaporated. The aqueous concentrate was taken up in DCM and water andthe mixture was extracted. The organic layer was separated, dried(MgSO₄), filtered and the solvent was evaporated till dryness. Theresidue was crystallized from 2-propanone and MeOH. The precipitate wasfiltered off, washed with diethyl ether and dried, yielding 1.2 g ofcompound 7, melting point 131° C.

Table F-1 lists the compounds that were prepared according to one of theabove Examples. The following abbreviations were used in the tables.

TABLE F-1

Co. No. 1; Ex. [B1]; mp. 204° C.

Co. No. 2; Ex. [B2]; mp. 211.4° C.

Co. No. 3; Ex. [B3]; mp. 180° C.

Co. No. 4; Ex. [B4]

Co. No. 5; Ex. [B5]

Co. No. 6; Ex. [B6]; mp. 221.3° C.

Co. No. 7; Ex. [B7]; mp. 131° C.

Co. No. 8; Ex. [B1]; mp. 163° C.

Co. No. 9; Ex. [B1]; mp. 215° C.

Co. No. 10; Ex. [B3]; mp. 125° C.

Co. No. 11; Ex. [B3]; mp. 100° C.

•HCl (1:2); Co. No. 12; Ex. [B3]; mp. >260° C.

•C₂H₂O₄ (2:5). H2O (1:1); Co. No. 13; Ex. [B3]; mp. 126° C.

C₂H₂O₄ (1:2); Co. No. 14; Ex. [B3]

Co. No. 15; Ex. [B5]

Co. No. 16; Ex. [B5]

Co. No. 17; Ex. [B5]; mp. 212° C.

Co. No. 18; Ex. [B6]; mp. 212.7° C.

Co. No. 19; Ex. [B7]; mp. 165° C.

EP0371564; Co. No. 20

EP0371564; Co. No. 21

EP0371564; Co. No. 22

EP0371564; Co. No. 23

EP0371564; Co. No. 24.

EP0371564; Co. No. 25

Pharmacological Example In Vitro Scintillation Proximity Assay (SPA) forPARP-1 Inhibitory Activity

Compounds of the present invention were tested in an in vitro assaybased on SPA technology (proprietary to Amersham Pharmacia Biotech).

In principle, the assay relies upon the well established SPA technologyfor the detection of poly(ADP-ribosyl)ation of biotinylated targetproteins, i.e histones. This ribosylation is induced using nicked DNAactivated PARP-1 enzyme and [³H]-nicotinamide adenine dinucleotide([³H]-NAD⁺) as ADP-ribosyl donor.

As inducer of PARP-1 enzyme activity, nicked DNA was prepared. For this,25 mg of DNA (supplier: Sigma) was dissolved in 25 ml DNAse buffer (10mM Tris-HCl, pH 7.4; 0.5 mg/ml Bovine Serum Albumine (BSA); 5 mMMgCl₂.6H₂O and 1 mM KCl) to which 50 μl DNAse solution (1 mg/ml in 0.15M NaCl) was added. After an incubation of 90 min. at 37° C., thereaction was terminated by adding 1.45 g NaCl, followed by a furtherincubation at 58° C. for 15 min. The reaction mixture was cooled on iceand dialysed at 4° C. for respectively 1.5 and 2 hours against 1.5 l of0.2 M KCl, and twice against 1.5 l of 0.01 M KCl for 1.5 and 2 hrespectively. The mixture was aliquoted and stored at −20° C. Histones(1 mg/ml, type II-A, supplier: Sigma) were biotinylated using thebiotinylation kit of Amersham and stored aliquoted at −20° C. A stocksolution of 100 mg/ml SPA poly(vinyl toluene) (PVT) beads (supplier:Amersham) was made in PBS. A stock solution of [³H]-NAD⁺ was made byadding 120 μl of [³H]-NAD⁺ (0.1 mCi/ml, supplier: NEN) to 6 mlincubation buffer (50 mM Tris/HCl, pH 8; 0.2 mM DTT; 4 mM MgCl₂). Asolution of 4 mM NAD⁺ (supplier: Roche) was made in incubation buffer(from a 100 mM stock solution in water stored at −20° C.). The PARP-1enzyme was produced using art known techniques, i.e. cloning andexpression of the protein starting from human liver cDNA. Informationconcerning the used protein sequence of the PARP-1 enzyme includingliterature references can be found in the Swiss-Prot database underprimary accession number P09874. Biotinylated histones and PVT-SPA beadswere mixed and pre-incubated for 30 min. at room temperature. PARP-1enzyme (concentration was lot dependent) was mixed with the nicked DNAand the mixture was pre-incubated for 30 min. at 4° C. Equal parts ofthis histones/PVT-SPA beads solution and PARP-1 enzyme/DNA solution weremixed and 75 μl of this mixture together with 1 μl of compound in DMSOand 25 μl of [³H]-NAD⁺ was added per well into a 96-wellmicrotiterplate. The final concentrations in the incubation mixture were2 μg/ml for the biotinylated histones, 2 mg/ml for the PVT-SPA beads, 2μg/ml for the nicked DNA and between 5-10 μg/ml for the PARP-1 enzyme.After incubation of the mixture for 15 min. at room temperature, thereaction was terminated by adding 100 μl of 4 mM NAD⁺ in incubationbuffer (final concentration 2 mM) and plates were mixed.

The beads were allowed to sediment for at least 15 min. and platestransferred to a TopCountNXT™ (Packard) for scintillation counting,values were expressed as counts per minute (cpm). For each experiment,controls (containing PARP-1 enzyme and DMSO without compound), a blankincubation (containing DMSO but no PARP-1 enzyme or compound) andsamples (containing PARP-1 enzyme and compound dissolved in DMSO) wererun in parallel. All compounds tested were dissolved and eventuallyfurther diluted in DMSO. In first instance, compounds were tested at aconcentration of 10⁻⁶ M. When the compounds showed activity at 10⁻⁶ M, adose-response curve was made wherein the compounds were tested atconcentrations between 10⁻⁵M and 10⁻⁸M. In each test, the blank valuewas subtracted from both the control and the sample values. The controlsample represented maximal PARP-1 enzyme activity. For each sample, theamount of cpm was expressed as a percentage of the mean cpm value of thecontrols. When appropriate, IC₅₀-values (concentration of the drug,needed to reduce the PARP-1 enzyme activity to 50% of the control) werecomputed using linear interpolation between the experimental points justabove and below the 50% level. Herein the effects of test compounds areexpressed as pIC₅₀ (the negative log value of the IC₅₀-value). As areference compound, 4-amino-1,8-naphthalimide was included to validatethe SPA assay. The tested compounds showed inhibitory activity at theinitial test concentration of 10⁻⁶ M (see Table-2).

In Vitro Filtration Assay for PARP-1 Inhibitory Activity

Compounds of the present invention were tested in an in vitro filtrationassay assessing PARP-1 activity (triggered in the presence of nickedDNA) by means of its histone poly(ADP-ribosyl)ation activity using[³²P]-NAD as ADP-ribosyl donor. The radioactive ribosylated histoneswere precipitated by trichloroacetic acid (TCA) in 96-well filterplatesand the incorporated [³²P] measured using a scintillation counter

A mixture of histones (stock solution: 5 mg/ml in H₂O), NAD⁺ (stocksolution: 100 mM in H₂O), and [³²P]-NAD⁺ in incubation buffer (50 mMTris/HCl, pH 8; 0.2 mM DTT; 4 mM MgCl₂) was made. A mixture of thePARP-1 enzyme (5-10 μg/ml) and nicked DNA was also made. The nicked DNAwas prepared as described in the in vitro SPA for PARP-1 inhibitoryactivity. Seventy-five μl of the PARP-1 enzyme/DNA mixture together with1 μl of compound in DMSO and 25 μl of histones-NAD⁺/[³²P]-NAD⁺ mixturewas added per well of a 96-well filterplate (0.45 μm, supplierMillipore). The final concentrations in the incubation mixture were2μg/ml for the histones, 0.1 mM for the NAD⁺, 200 μM (0.5 μC) for the[³²P]-NAD⁺ and 2μg/ml for the nicked DNA. Plates were incubated for 15min. at room temperature and the reaction was terminated by the additionof 10 μl ice cold 100% TCA followed by the addition of 10 μl ice-coldBSA solution (1% in H₂O). The protein fraction was allowed toprecipitate for 10 min. at 4° C. and plates were vacuum filtered . Theplates were subsequently washed with, for each well, 1 ml of 10% icecold TCA, 1 ml of 5% ice cold TCA and 1 ml of 5% TCA at roomtemperature. Finally 100 μl of scintillation solution (Microscint 40,Packard) was added to each well and the plates were transferred to aTopCountNXT™ (supplier: Packard) for scintillation counting and valueswere expressed as counts per minute (cpm). For each experiment, controls(containing PARP-1 enzyme and DMSO without compound), a blank incubation(containing DMSO but no PARP-1 enzyme or compound) and samples(containing PARP-1 enzyme and compound dissolved in DMSO) were run inparallel. All compounds tested were dissolved and eventually furtherdiluted in DMSO. In first instance, compounds were tested at aconcentration of 10⁻⁵M. When the compounds showed activity at 10⁻⁵M, adose-response curve was made wherein the compounds were tested atconcentrations between 10⁻⁵M and 10⁻⁸M. In each test, the blank valuewas subtracted from both the control and the sample values. The controlsample represented maximal PARP-1 enzyme activity. For each sample, theamount of cpm was expressed as a percentage of the mean cpm value of thecontrols. When appropriate, IC₅₀-values (concentration of the drug,needed to reduce the PARP-1 enzyme activity to 50% of the control) werecomputed using linear interpolation between the experimental points justabove and below the 50% level. Herein the effects of test compounds areexpressed as pIC_(5o) (the negative log value of the IC₅₀-value). As areference compound, 4-amino-1,8-naphthalimide was included to validatethe filtration assay. The tested compounds showed inhibitory activity atthe initial test concentration of 10⁻⁵M (see Table-2).

TABLE 2 In vitro In vitro SPA filtration assay Co No pIC50 pIC50 1 6.6562 6.282 5.272 3 6.587 5.586 4 5.983 5.121 5 6.807 6.195 6 6.114 5 76.674 6.112 8 6.011 9 6.129 10 6.131 11 6.485 12 6.163 13 6.434 14 6.30215 5.901 5.441 16 6.328 5.665 17 6.704 5.834 18 5.99 5.196 19 6.127 206.359 5.642 21 6.644 5.958 22 6.077 5.364 23 5.844 5.147 24 6.251 5.31325 6 5.334

The compounds can be further evaluated in a cellular chemo- and/orradiosensitization assay, an assay measuring inhibition of endogenousPARP-1 activity in cancer cell lines and eventually in an in vivoradiosensitization test.

1. A compound of formula (I),

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein n is 0, 1 or 2; Xis N or CR⁵, wherein R⁵ is hydrogen or taken together with R¹ may form abivalent radical of formula —CH═CH—CH═CH—; R¹ is C₁₋₆alkyl or thienyl;R² is hydrogen or hydroxy or taken together with R³ or R⁴ may form ═O;R³ is a radical selected from—(CH₂)_(s)—NR⁶R⁷  (a-1),—O—H  (a-2),—O—R⁸  (a-3),—S—R⁹  (a-4), or—C≡N  (a-5), wherein s is 0, 1, 2 or 3; R⁶ is —CHO, C₁₋₆alkyl,hydroxyC₁₋₆alkyl, C₁₋₆alkylcarbonyl, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkylcarbonylaminoC₁₋₆alkyl,piperidinylC₁₋₆alkylaminocarbonyl, piperidinyl, piperidinylC₁₋₆alkyl,piperidinylC₁₋₆alkylaminocarbonyl, C₁₋₆alkyloxy, thienylC₁₋₆alkyl,pyrrolylC₁₋₆alkyl, arylC₁₋₆alkylpiperidinyl, arylcarbonylC₁₋₆alkyl,arylcarbonylpiperidinylC₁₋₆alkyl, haloindozolylpiperidinylC₁₋₆alkyl, orarylC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl; R⁷ is hydrogen or C₁₋₆alkyl; R⁸is C₁₋₆alkyl, C₁₋₆alkylcarbonyl or di(C₁₋₆alkyl)aminoC₁₋₆alkyl; and R⁹is di(C₁₋₆alkyl)aminoC₁₋₆alkyl; or R³ is a group of formula—Z—  (b-1), wherein Z is a heterocyclic ring system selected from

wherein each R¹⁰ independently is hydrogen, C₁₋₆alkyl, aminocarbonyl,hydroxy,

C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkylamino, arylC₁₋₆alkyl,di(phenylC₂₋₆alkenyl), piperidinylC₁₋₆alkyl, C₃₋₁₀cycloalkyl,C₃₋₁₀cycloalkylC₁₋₆alkyl, aryloxy(hydroxy)C₁₋₆alkyl, haloindazolyl,arylC₁₋₆alkyl, arylC₂₋₆alkenyl, morpholino, C₁₋₆alkylimidazolyl, orpyridinylC₁₋₆alkylamino; R⁴ is hydrogen, C₁₋₆alkyl, furanyl, pyridinyl,arylC₁₋₆alkyl or

aryl is phenyl or phenyl substituted with halo, C₁₋₆alkyl orC₁₋₆alkyloxy; with the proviso that when n is 0, X is N, R² is hydrogen,R³ is a group of formula (b-1), Z is the heterocyclic ring system (c-2)or (c-4) wherein said heterocyclic ring system Z is attached to the restof the molecule with a nitrogen atom, and R¹⁰ is hydrogen; then R⁴ isother than C₁₋₆alkyl or pyridinyl.
 2. A compound as claimed in claim 1wherein n is 0 or 1; X is N or CR⁵, wherein R⁵ is hydrogen; R³ is aradical selected from (a-1), (a-2) or (a-3) or is a group of formula(b-1) i.e. —Z—; s is 0, 1 or 2; R⁶ is —CHO, C₁₋₆alkyl,piperidinylC₁₋₆alkyl, arylcarbonylpiperidinylC₁₋₆alkyl orarylC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl; R⁸ is C₁₋₆alkyl; when R³ is agroup of formula (b-1) then Z is a heterocyclic ring system selectedfrom (c-2) or (c-4); and each R¹⁰ independently is hydrogen, C₁₋₆alkylor C₁₋₆alkyloxyC₁₋₆alkylamino
 3. (canceled)
 4. (canceled)
 5. (canceled)6. A pharmaceutical composition comprising pharmaceutically acceptablecarriers and as an active ingredient a therapeutically effective amountof a compound as claimed in claim
 1. 7. A process of preparing apharmaceutical composition as claimed in claim 6 wherein thepharmaceutically acceptable carriers and a compound as claimed in claim1 are intimately mixed.
 8. Use of a compound for the manufacture of amedicament for the treatment of a PARP mediated disorder, wherein saidcompound is a compound of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein n is 0, 1 or 2; Xis N or CR⁵, wherein R⁵ is hydrogen or taken together with R¹ may form abivalent radical of formula —CH═CH—CH═CH—; R¹ is C₁₋₆alkyl or thienyl;R² is hydrogen or hydroxy or taken together with R³ or R⁴ may form ═O;R³ is a radical selected from—(CH₂)_(s)—NR⁶R⁷  (a-1),—O—H  (a-2),—O—R⁸  (a-3),—S—R⁹  (a-4), or—C≡N  (a-5), wherein s is 0, 1, 2 or 3; R⁶ is —CHO, C₁₋₆alkyl,hydroxyC₁₋₆alkyl, C₁₋₆alkylcarbonyl, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkylcarbonylaminoC₁₋₆alkyl,piperidinylC₁₋₆alkylaminocarbonyl, piperidinyl, piperidinylC₁₋₆alkyl,piperidinylC₁₋₆alkylaminocarbonyl, C₁₋₆alkyloxy, thienylC₁₋₆alkyl,pyrrolylC₁₋₆alkyl, arylC₁₋₆alkylpiperidinyl, arylcarbonylC₁₋₆alkyl,arylcarbonylpiperidinylC₁₋₆alkyl, haloindozolylpiperidinylC₁₋₆alkyl, orarylC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl; R⁷ is hydrogen or C₁₋₆alkyl; R⁸is C₁₋₆alkyl, C₁₋₆alkylcarbonyl or di(C₁₋₆alkyl)aminoC₁₋₆alkyl; and R⁹is di(C₁₋₆alkyl)aminoC₁₋₆alkyl; or R³ is a group of formula—Z—  (b-1), wherein Z is a heterocyclic ring system selected from

wherein each R¹⁰ independently is hydrogen, C₁₋₆alkyl, aminocarbonyl,hydroxy,

C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkylamino, arylC₁₋₆alkyl,di(phenylC₂₋₆alkenyl), piperidinylC₁₋₆alkyl, C₃₋₁₀cycloalkyl,C₃₋₁₀cycloalkylC₁₋₆alkyl, aryloxy(hydroxy)C₁₋₆alkyl, haloindazolyl,arylC₁₋₆alkyl, arylC₂₋₆alkenyl, morpholino, C₁₋₆alkylimidazolyl, orpyridinylC₁₋₆alkylamino; R⁴ is hydrogen, C₁₋₆alkyl, furanyl, pyridinyl,arylC₁₋₆alkyl or

aryl is phenyl or phenyl substituted with halo, C₁₋₆alkyl orC₁₋₆alkyloxy.
 9. Use according to claim 8 of a PARP inhibitor of formula(I) for the manufacture of a medicament for the treatment of a PARP-1mediated disorder
 10. Use according to claims 8 and 9 wherein thetreatment involves chemosensitization.
 11. Use according to claims 8 and9 wherein the treatment involves radiosensitization.
 12. A combinationof a compound with a chemotherapeutic agent wherein said compound is acompound of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein n is 0, 1 or 2; Xis N or CR⁵, wherein R⁵ is hydrogen or taken together with R¹ may form abivalent radical of formula —CH═CH—CH═CH—; R¹ is C₁₋₆alkyl or thienyl;R² is hydrogen or hydroxy or taken together with R³ or R⁴ may form ═O;R³ is a radical selected from—(CH₂)_(s)—NR⁶R⁷  (a-1),—O—H  (a-2),—O—R⁸  (a-3),—S—R⁹  (a-4), or—C≡N  (a-5), wherein s is 0, 1, 2 or 3; R⁶ is —CHO, C₁₋₆alkyl,hydroxyC₁₋₆alkyl, C₁₋₆alkylcarbonyl, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkylcarbonylaminoC₁₋₆alkyl,piperidinylC₁₋₆alkylaminocarbonyl, piperidinyl, piperidinylC₁₋₆alkyl,piperidinylC₁₋₆alkylaminocarbonyl, C₁₋₆alkyloxy, thienylC₁₋₆alkyl,pyrrolylC₁₋₆alkyl, arylC₁₋₆alkylpiperidinyl, arylcarbonylC₁₋₆ alkyl,arylcarbonylpiperidinylC₁₋₆alkyl, haloindozolylpiperidinylC₁₋₆alkyl, orarylC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl; R⁷ is hydrogen or C₁₋₆alkyl; R⁸is C₁₋₆alkyl, C₁₋₆alkylcarbonyl or di(C₁₋₆alkyl)aminoC₁₋₆alkyl; and R⁹is di(C₁₋₆alkyl)aminoC₁₋₆alkyl; or R³ is a group of formula—Z—  (b-1), wherein Z is a heterocyclic ring system selected from

wherein each R¹⁰ independently is hydrogen, C₁₋₆alkyl, aminocarbonyl,hydroxy,

C₁₋₆alkyloxyC₁₋₆alkyl, C₁₋₆alkyloxyC₁₋₆alkylamino, arylC₁₋₆alkyl,di(phenylC₂₋₆alkenyl), piperidinylC₁₋₆alkyl, C₃₋₁₀cycloalkyl,C₃₋₁₀cycloalkylC₁₋₆alkyl, aryloxy(hydroxy)C₁₋₆alkyl, haloindazolyl,arylC₁₋₆alkyl, arylC₂₋₆alkenyl, morpholino, C₁₋₆alkylimidazolyl, orpyridinylC₁₋₆alkylamino; R⁴ is hydrogen, C₁₋₆alkyl, furanyl, pyridinyl,arylC₁₋₆alkyl or

aryl is phenyl or phenyl substituted with halo, C₁₋₆alkyl orC₁₋₆alkyloxy.
 13. (canceled)
 14. A pharmaceutical composition comprisingpharmaceutically acceptable carriers and as an active ingredient atherapeutically effective amount of a compound as claimed in claim 2.15. (canceled)
 16. (canceled)
 17. A method of treating in a subject aPARP mediated disorder, said method comprising administering to thesubject a therapeutically effective amount of a compound of claim
 2. 18.A method for enhancing the effectiveness of chemotherapy comprisingadministration of a compound according to claim 2, in a therapeuticallyeffective amount so as to increase sensitivity of cells to chemotherapy,prior to administration of said chemotherapy .
 19. A method forenhancing the effectiveness of radiotherapy comprising administration ofa compound according to claim 2, in a therapeutically effective amountso as to increase sensitivity of cells to ionizing radiation, prior toadministration of said radiotherapy. 20.-25. (canceled)
 26. Acombination of a compound with a chemotherapeutic agent wherein saidcompound is a compound of claim
 2. 27. (canceled)
 28. (canceled)
 29. Aproduct made by the process of claim
 13. 30. A pharmaceuticalcomposition made by the process of claim
 13. 31. (canceled) 32.(canceled)
 33. (canceled)